Review Article In vivo molecular imaging of cancer stem cells

Am J Nucl Med Mol Imaging 2015;5(1):14-26 www.ajnmmi.us /ISSN:2160-8407/ajnmmi0002123

Review Article In vivo molecular imaging of cancer stem cells Yusri D Heryanto1, Arifudin Achmad1,2,3, Ayako Taketomi-Takahashi1, Yoshito Tsushima1 Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; 2Human Research Cultivation Center, Gunma University, Kiryu, Gunma, Japan; 3Department of Radiology, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia 1

Received August 27, 2014; Accepted September 5, 2014; Epub December 15, 2014; Published January 1, 2015 Abstract: A rare subpopulation of cancer cells known as cancer stem cells (CSCs) have distinct characteristics resembling stem cells, including cell renewal capability, differentiation into multiple lineages, and endless proliferation potential. Cumulating evidence has revealed that CSCs are responsible for tumorigenicity, invasion, metastasis, and therapeutic resistance. Despite continued investigation of CSCs, in vivo behavior of CSCs is not yet fully understood. The in vivo imaging modalities of optical, nuclear, and magnetic resonance are currently being employed to investigate the complexity behind the CSCs behavior. Valuable information that were previously obscured by the limitations of in vitro techniques now are currently being revealed. These studies give us a more comprehensive insight about what happen to CSCs in vivo. This review will briefly discuss the recent findings on CSCs behavior as informed by in vivo imaging studies. Keywords: Cancer stem cell, MRI, PET, fluorescence imaging, in vivo imaging, molecular imaging, cell tracking

Introduction Tumors consist of both tumor cells and those that make up the tumor microenvironment. Tumor cells are composed of heterogeneous subpopulations with distinct genotypes and phenotypes, which may harbor divergent biological behaviors [1]. Recently, there is accumulating evidence on the existence of a tumor subpopulation termed cancer stem cell (CSCs) with distinct characteristics resembling stem cells [2]. This CSCs hypothesis has become one of the most intriguing and challenging concepts in oncology. This hypothesis states that the initiation and growth of cancer is controlled by a small subpopulation that has stem cells-like characteristics. Eradication of the stem-cell compartment of a tumor may be essential to achieve stable, long-lasting remission, and cure of cancer [3]. Many studies have been performed over the past 40 to 50 years to learn about CSCs [4-8]; however, the complex behavior of CSCs in vivo remains largely a mystery. Nowadays, in vivo imaging technologies provide opportunities to deepen our understanding about CSC characteristics. Advances in experimental and clinical

imaging enable researchers and physicians not only to locate the CSCs but also to assess the tumor biological processes involving CSCs. This review will briefly discuss in vivo imaging modalities and techniques that have been used to visualize and track CSCs. Cancer stem cells CSCs are defined by The American Association for Cancer Research Stem Cell Workshop as cells within a tumor that possess the capacity to self-renew and to inherit heterogeneous lineages of cancer cells that comprise the tumor [9]. Compared to the other tumor subpopulations, CSCs are unique due to three distinctive characteristics (Table 1): the capacity of selfrenewal (i.e., one or both daughter cells retain the same biologic properties as the parent cell at cell division), the capability to develop into multiple lineages, and the potential to proliferate extensively [2]. The first evidence of CSCs existence was found 50 years ago. A single tumor cell derived from ascites fluid of teratocarcinomas and leukemia had tumorigenic potential and could generate heterogeneous progeny [5, 8, 10]. Later, CSCs

Imaging of cancer stem cells Table 1. Characteristics of CSCs Characteristics of CSCs Capacity of self-renewal Capability to develop into multiple lineages Potential to proliferate extensively Rare subpopulation of cells Radioresistance Chemoresistance Promote invasion and metastatic activity

Reference [4, 6, 7, 14] [4, 6, 7, 14] [4, 6, 7, 14] [4] [22, 23] [19-21] [26-28]

Table 2. CSCs surface biomarker Tumor entity Breast cancer Glioblastoma Prostate cancer Melanoma Lung cancer Liver cancer Colon cancer Pancreatic cancer Head and neck cancer Acute myeloid leukemia Multiple myeloma

Markers CD44+CD24-/low CD133+ CD133+ CD44+/α2β1hi/CD133+ CD20+ CD133+ CD44 high CD90+ CD133+ CD90+CD44+ CD133+ EpCAM+CD44+CD166+ CD44+CD24+ESA+ CD133+CXCR4+ CD44+ CD34+CD38+ CD138+

were found in hematological tumor. Injection of a small subset of leukemic cells with a primitive hematopoietic progenitor phenotype (CD34+ CD38-) into immunodeficient mice gave them Acute Myeloid Leukemia (AML). The frequency of this subset was found to be rare (± one per million tumor cells) [4]. Since then, many studies were performed to identify and isolate CSCs subpopulations in other tumor types including brain, breast, colon, pancreas, prostate, lung, and head and neck cancer [6, 7, 11-13]. CSCs may come from normal tissue stem cells (TSCs) that undergo mutation. TSCs, which proliferate throughout life, are more susceptible to oncogenic mutations. Moreover, TSC markers and differentiation markers such as CD133 and CD44 have been found in many cancers [14, 15]. Also, stem cells and precursor cells transformed with oncogenic genes can develop cancer in vivo [16]. However, several experiments showed that CSCs might also arise from 15

Ref. [14] [73] [15] [6] [74] [75] [76] [77] [78] [79] [7] [12] [27] [13] [42] [41]

more committed progenitor cells that regained self-renewal properties [16-18]. CSCs are less sensitive to conventional therapies such as chemotherapy [19-21] and radiotherapy [22, 23]. This resistance is due to factors such as the quiescent state of CSCs, enhanced DNA repair, upregulated cell cycle control mechanisms, reactive oxygen species scavenging, and interaction with stromal environment. The detailed mechanisms of radioresistance and chemoresistance are well described in other reviews [24, 25]. CSCs also promote invasion and metastatic activity [26-28]. The initial approach in CSC investigation is isolation and identification. Assays for CSC activity should focus on demonstrating both self-renewal and tumor propagation, because those two properties are the hallmarks of CSC. The gold standard assay that fulfills these criteria is serial transplantation in animal models. Unfortunately, this method is tedious and time consuming [9]. To overcome those limitations, simpler methods have been devised such as cell side population (SP) exclusion [29-32], floating sphere formation [20, 33-36], and aldehyde dehydrogenase (ALDH) activity assay [37-40]. Another important method used for the identification of CSCs is based on knowledge derived from hematopoietic or embryonic stem cell development (Table 2). For example, CD138, a marker for terminally differentiated B cells, is negative in multiple myeloma (a plasma cell malignancy) CSCs [41]. Likewise, CSC markers from acute myelogenous leukemia are CD34+CD38- which are the same markers used to identify normal early hematopoietic progenitor cells [42]. Surface markers allow for the definition of a precise population. However, they may not be sufficiently reliable in discriminating cancer stem cells from non-stem cell subpopulations. Another limitation is that markers derived from cell-line studies or mouse tumors have yet to be validated in human tumors [43, 44]. Imaging cancer stem cell Advances in imaging technology allow us to explore the biology of CSCs. Some of these modalities are promising as clinical applications. Modalities used for in vivo imaging of Am J Nucl Med Mol Imaging 2015;5(1):14-26

Imaging of cancer stem cells Table 3. Advantages and limitations of imaging modality for CSC study [46, 48, 80] Imaging modality

Resolution1

Depth

Time for image acquisition

Quantitative2

Target

MRI

10-100 µm

no limit

minutes to hours

yes

Anatomical, physiological, molecular

yes

Non-invasive, non-radioactive, excellent spatial resolution but costly

CT

50 µm

no limit

minutes

yes

Anatomical, physiological,

yes

Usually combined with PET or SPECT, high radiation exposure

PET/SPECT

1-2 mm

no limit

minutes to hours

yes

Physiological, molecular

yes

Versatile imaging modality with many tracers and high sensitivity

Fluorescence reflectance imaging

2-3 mm

< 1 cm

seconds to minutes

no


yes

Suitable for imaging molecular events at surface tumor but have limited depth resolution

FMT

1 mm

< 10 cm

minutes to hours

yes


in development

Quantitative imaging of fluorochrome reporters

Bioluminescence imaging

several mm

cm

minutes

no

Molecular

no

Can detect gene expression rare cells such CSC at high resolution

Intravital microscopy3

1 µm

< 400800 µm

seconds to hours

no

Anatomical, physiological, molecular

in development

Clinical Notes use

Single cell resolution but have limited depths and coverage for CSC study

High resolution for small animal imaging. 2Quantitative here means inherently quantitative. All approaches allow relative quantification. 3Laser-scanning confocal or multiphoton microscopy. 1

CSCs include radionuclide imaging such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), intravital imaging, bioluminescence imaging, and various fluorescence imaging including fluorescence-mediated tomography (FMT) and near infrared (NIR) fluorescence reflectance imaging. In case of in vivo CSCs imaging, there is no “best” imaging modality. The choice of specific imaging modality is depend on the purpose of the study. For investigating therapeutic response or metastatic CSCs, PET and MRI are the most suitable modality. Whereas optical imaging modality is favorable for investigating tumor propagation and plasticity of CSCs with single cell resolution. In Table 3, we summarized each of imaging modality’s strengths and limitations. In CSCs imaging, we need to choose the specific imaging modality, the reporter gene or probe, the imaging requirement (single versus repeated acquisition), intended use (animal versus human), and spatial requirements (organ versus cellular resolution) [45]. Because CSCs are rare subpopulation of cells, the imaging modality must be sensitive to the contrast agent at a resolution that can detect cells with frequency as low as one in one thousand [46]. Optical imaging Optical imaging is unequivocally the most versatile and widely used modality in clinical prac16

tice and research. In addition, optical imaging techniques are the easiest to apply to CSCs study at the resolution of a single cell, and relatively cheaper than other imaging modalities [46] . As CSCs are a rare subpopulation of cells, the main concerns in optical imaging are choosing reporter signal and imaging modalities. Bioluminescence signal can define tumor growth, regression, and metastases but its spatial resolution and sensitivity in identifying rare cells is limited [46]. Luciferase reporter plasmid is highly sensitive for measuring biologic activity in growing tumor, but in luciferase models, animals must be injected with the luciferin substrate. Detection of signal in vivo requires expression of luciferase in at least 2500 cells [47]. Fluorescence imaging is currently the best choice for imaging CSCs. Sensitive detectors and the intensity and stability of fluorescence signal allow imaging of fluorescent cells in vivo at high resolutions. Multiple fluorophores can be used at the same time, which is useful in imaging the complex biological features of CSCs. Factors that need to be considered in choosing optical imaging devices for studying CSCs are depth of tissue penetration, imaging time points, and use of multispectral unmixing [46]. Depth of penetration is important in studying and imaging internal organs or subcutaneous xenograft models. Near-infrared fluorescence (NIR) probes have the highest penetration. Am J Nucl Med Mol Imaging 2015;5(1):14-26

Imaging of cancer stem cells

Figure 1. In vivo multiphoton microscopy from experiments depicted in scheme (A) followed up the tumor growth from cancer stem cells. Projection micrographs (B-D) demonstrate tumor formation over time and three-dimensional

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Am J Nucl Med Mol Imaging 2015;5(1):14-26

Imaging of cancer stem cells reconstructions depicted in micrographs (E, E’) revealed CSCs (white arrows) was associated with blood vessels and and grew in proximity with blood vessels (D, E). Over time (from day 13 to 20), a tumor nodule rapidly formed and tumor cells (blue arrows) were observed to infiltrate the peripheral regions (D, E). Fluorescent dextran (shown in red) was injected into the circulation to illuminate blood vessels prior to imaging. Scale bar represents 50 µm [51].

Figure 2. Fractionated CSCs and non-stem tumor cells were labeled with different fluorescent proteins and transplanted into mice at a 10% cancer stem cell (YFP) to 90% non-stem tumor cell (CFP) ratio as shown in experimental

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Imaging of cancer stem cells design schematic (A). Graph (B) which is calculated based on three-dimensional reconstructions of projection micrographs (C, D) demonstrated that CSCs outgrew non-CSCs in vivo. Additionally, tumor populations did not intermingle in vivo (non-stem tumor population indicated by yellow oval). Fluorescent dextran (shown in purple) was injected into the circulation to illuminate blood vessels prior to imaging. Scale bar represents 100 µm [51].

Surgical manipulation such as skin flaps can also enhance penetration of probes. Multiple imaging time points are necessary in longitudinal studies. Spectral unmixing removes autofluorescence and can distinguish between emission peaks of nominal separation [48]. There have been studies imaging CSCs using optical imaging. Tsurumi et al showed in vivo detection of CD133, a cancer stem cell surface marker for a variety of tumor entities. They labeled AC133.1, a CD133-specific monoclonal antibody (mAb), with fluorescence dye for quantitative NIR fluorescence molecular tomography (NIR FMT) imaging on mouse subcutaneous xenograft models. They visualized and quantified CD133 in lentivirally-transduced CD133 overexpressing cells lines. Cells that express CD133 at endogenous levels had lower signal to noise ratio [49]. Gaedicke et al continued the Tsurumi et al study by showing that in vivo CD133 imaging using fluorescence labelled-mAb could also be applied to an orthotopic glioblastoma model. The fluorescence signal of ACC133 mAb was significantly higher in the tumor region than in the isotype control antibody. This study revealed that systemically administered antibodies may reach extravascular targets in brain tumors with a disturbed blood brain barrier, and also showed the potential of clinical translation of CD133 imaging [50]. Intravital microscopy techniques can be used study the behavior of cells by visualizing tissues in living animals. Advances in intravital microscopy enable visualization of CSCs at a resolution of up to 1 µm, allowing single cell resolution imaging. Additional developments are deeper penetration, minimal image distortion, signal quantification, and three-dimensional image reconstruction [51, 52]. Using intravital microscopy, Lathia et al depicted CSCs growth in vivo over a temporal time course using lentivirus-transduced fluorescence-labeled CSCs (CD133+ cells). As the first intravital imaging study using multiphoton microscopy to observe tumor growth from

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CSCs, this study showed how CSCs flourished from a perivascular niche and outgrew the nonCSCs cells over a temporal time course (Figures 1 and 2) [51]. Stem cells-like properties may not be permanent. CSCs may lose stemness properties, and non-CSCs may gain them. This phenomenon is known as stem cell plasticity [53, 54]. Intravital microscopy has been used to observe cancer stem cell plasticity. Zomer et al proved this hypothesis by intravital lineage tracing. They lineage traced the growth of genetic mammary tumors that expressed a tamoxifen-activated Cre confetti construct. In this mouse models, Cre activation induced random expression of one of four confetti fluorescent colors (cyan, green, yellow, and red). Cre-expressing cells and their progeny would be genetically marked by one of the confetti colors, which allowed lineage tracing of these cells. They observed that only some of the cell grew and formed single colored patches four days after being activated. This showed that a tumor population developed from a small tumor subpopulation. Several days later, other new patches appeared, while some of the previous patches regressed, suggested that CSC properties were occasionally acquired and may also be lost [55]. A new NIR-sensitive molecular imaging probe based on hydrogel complexes can visualize CD44-expressing cancer stem cells in gastric cancer. The probe, NIR-sensitive supramolecular hydrogels (Cy 5.5-conjugated polyethyleneimine/hyaluronic acidpolyplexes), was fabricated by polyplexing in an aqueous medium. This probe demonstrated good water-stability, biocompatibility, and specificity to CD44 [56]. Another approach to study CSC behavior in vivo is by using reporter genes. Liu et al applied dual-function bioluminescence imaging-fluorescent reporter constructs (Luc2 fused with eGFP coding sequence) in breast cancer stem cells (BCSCs). Luc2 sequence was employed for whole body tracking by bioluminescence imaging (BLI) while eGFP was used for intravital imaging and ex vivo analysis. These optical reporters stably expressed over series of tumor



Figure 3. Follow-up volumetric MRI images of BCSC, BCSC treated with docetaxel (BCSC Doc), FTH-BCSC and FTHBCSC treated with docetaxel (FTH-BCSC Doc). After docetaxel treatment, the mean R2* values of the BCSC Doc and FTH-BCSC Doc tumors were significantly decreased compared with the BCSC and FTH-BCSC tumors at days 5 and days 14. Moreover MRI images, revealed significantly different signal intensities (R2* values) between FTH genestransduced BCSCs (FTH-BCSC & FTH-BCSC Doc) and wild type BCSCs (BCSC & BCSC Doc) [69].

progeny. Using BLI, as few as ten injected CD44+ BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis [57]. Besides surface protein markers, proteasome activity can also be used for in vivo tracking of CSCs. Vlashi et al found that 26S proteasome activity was reduced in CSCs. Using this finding, they performed in vivo CSCs tracking in human glioma and breast cancer. They engineered cancer cells to stably express fluorescence fusion protein (ZsGreen-ornithine decarboxylase) that accumulates in cells that have reduced 26S proteasome activity. ZsGreenpositive cells were successfully tracked by in vivo fluorescence imaging. Compared with the ZsGreen-negative population, secondary sphere-forming capacity and in vivo tumorigenicity of ZsGreen-positive cells were significantly higher. Furthermore, ZsGreen-high cells were positive for the stem cell marker nestin, Musashi-1, and Sox2 and negative for differentiation markers such as GFAP and neuron-specific class III β-tubulin in vitro. These findings suggest that ZsGreen-high cells are CSCs [58]. Positron emission tomography (PET) PET quantitatively detects high-energy γ-rays emitted from a subject injected with positronemitting isotopes or isotope-labeled molecular probes. PET is highly sensitive, non-invasive, permits real time tracking in vivo, and independent of the depth from which the signal is emit-

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ted. However, until now, there is no PET technology capable of detecting CSC at single cell resolutions. Jin et al investigated the potential of in vivo radionuclide imaging of CSCs using Iodine-125labeled ANC9C5, an anti-human CD133 antibody, in colon carcinoma xenografts. Although a favorable biodistribution profile was not obtained, intratumoral distribution of 125I-labeled ANC9C5 depicted on autoradiography was overlapped with CD133 immunohistochemistry expression in many areas [59]. This study revealed the potential of radioimmunotargeting of CSCs using PET or SPECT. Gaedicke et al performed PET imaging to detect AC133, an epitope of the second extracellular loop of CD133. In this study, two cell lines overexpressing CD133 (engineered and patientderived) were xenografted in mice and imaged using 64Cu-NOTA-AC133. The imaging yielded accurate and high-resolution images of brain tumor lesions 2-3 mm in size, with significant tumor-to-background contrast. This study disclosed the differences in invasive behavior between orthotopically growing U251 (noninvasive) and NCH421k (invasive) gliomas. More interestingly, PET signal intensity accurately reflected the microscopic pattern of tumor AC133+ expression. This was observed in sharply delineated PET images of U251 tumor that has compact and spherical microscopic appearance. On the other hand, the chaotic and infiltrative growth pattern in NCH421k



Figure 4. Histological analysis with H&E staining of the area in the red and black boxes of MRI images on FTH genes-transduced BCSCs (FTH-BCSC & FTH-BCSC Doc) and wild type BCSCs (BCSC & BCSC Doc). High R2* value pixel showed the area of viable cells and the FTH gene enhances the contrast in MRI images by further increased the R2* values (A-D). Similar distribution of R2* value pixels in MRI between the periphery and center of both BCSC and FTH-BCSC were confirmed with H&E staining; FTH genes transduction enhanced the R2* values (A & C). After Docetaxel therapy, non viable cells were appeared in the center of the tumors (demarcated with black dotted line) (B & D). Without FTH genes transduction, different R2* value pixel in MRI (demarcated with white dotted line) failed to represent cell viability as shown by H&E staining (B). In FTH-BCSC cells, R2* value pixel correspond well with cell viability in both periphery and center of the tumor (D). Scale bar in H&E and MRI represents 100 µm and 500 µm respectively [69].

tumors was represented by more diffuse PET signal [50]. Magnetic resonance imaging (MRI) From pre-clinical and clinical point of view, MRI is a non-invasive and robust imaging modality 21

with high spatial resolution. MRI does not rely on radioactive isotopes, which may be an important advantage for longitudinal studies. Moreover, MRI can collect not only morphological but also pathophysiological information in living objects. The ability of MRI with contrast Am J Nucl Med Mol Imaging 2015;5(1):14-26

Imaging of cancer stem cells agents for labeling cells providing dynamic assessment of cell migration into target tissues [60]. MRI has been used to detect single cells in small animals in a wide range of studies extending from stem cells to cancer cell tracking [61]. To pinpoint a cell of interest from background signal, a label capable of producing significant positive or negative contrast on the appropriate pulse sequence is an absolute must. In addition to strong signal-altering properties (T1, T2 relaxation), an “ideal” label should have characteristics such as biocompatibility, lack of genetic modification or perturbation to the target cell, capability of single-cell detection, ability to image target cells over a long period, and ability to quantify cell number at a given locus [62]. Most of the magnetic resonance labels currently used in cell tracking are ultrasmall superparamagnetic iron oxide (USPIO) or superparamagnetic iron oxide (SPIO). Advantages of these contrast agents over conventional paramagnetic gadolinium-based contrast agents are low toxicity, subnanomolar-range detection limits, and higher contrast enhancement [6365]. SPIO has successfully detected and tracked transplanted human hepatic stem cells in vivo and also glioblastoma CSCs in vitro [66, 67]. In MRI, the amount of USPIO and SPIO, not the number of cells, determines signal change. Cell proliferation distributes iron into daughter cells, so the signal from individual cells decreases with each generation. Iron released from apoptotic or lytic cells can be internalized by macrophages in nearby tissue nearby, resulting in signal wrongly associated with target cells [68]. Choi et al used the MRI reporter gene ferritin to overcome these limitations for detecting human breast cancer stem cells (BCSCs). The overexpression of ferritin enabled cells to uptake more iron, producing low signal intensities in MRI [69]. As MRI reporters are stably expressed, even during cell division, they can be used for studying dynamic processes, for, example, the migration and invasion of cells of interest over an extended period. This can provide temporal and spatial information for anticancer treatment effects on a specific cell population. Quantification of the number of viable cancer cells in deep tissues can be monitored

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by calculating R2* ( = 1/T2*) values from T2* mapping of MRI images [70, 71]. Choi et al utilized dual reporter gene (human ferritin heavy chain [FTH] and enhanced green fluorescence protein [eGFP]) transducedBCSCs transplanted into NOD/SCID mice to allow noninvasive tracking of BCSC-derived populations and to show viable cell populations of tumors after docetaxel chemotherapy. MRI revealed significantly different signal intensities (R2* values) between BCSCs and FTHBCSCs in vitro and in vivo (Figure 3). Histological analysis revealed that areas showing high R2* values in docetaxel-treated FTH-BCSCs tumors by MRI contained more viable cell populations with high percentages of BCSCs (Figure 4). This experimental model system can be used to investigate the best treatments of BCSCsderived tumors [69]. Another labelling technique targeting cancer stem cells has also been developed. Lim et al designed hyaluronan-modified magnetic nanoclusters (HA-MNCs) for in vitro and in vivo detection of CD44-overexpressing breast cancer using a MR imaging. Hyaluronan, an immune-neutral polysaccharide, is a ligand for CD44. Biocompatible magnetic nanoclusters (MNC) of less than 100 nm in size is the optimum vehicle for this ligand since it may hinder reticuloendothelial trap to prolong its circulation and at the same time ensure tumor accumulation via enhanced permeation and retention effect. Hence, HA-MNCs exhibited excellent targeting efficiency to CD44-overexpressing cancer cells. Signal intensity on T2-weighted images was decreased in HA-MNCs-treated mice, demonstrating a remarkably high MR contrast effect. It can be concluded that HA-MNCs has high potential to image cancer stem cells in vivo and promising for diverse applications in tumor diagnosis [72]. Conclusion Even though CSCs have been investigated extensively, their in vivo behavior is still unclear. In vitro studies may not sufficiently depict the complexity of CSC biology. In vivo imaging techniques allow CSCs tracking and obtaining valuable information such as tumor development from CSC populations, stem cell plasticity, and their interaction with the surrounding environ-


Imaging of cancer stem cells ment. These techniques may be used to investigate yet unexplored behavior such as interaction with the immune system, behavior under hypoxic conditions and stem cell-like property inheritance. CSCs are also responsible for therapeutic resistance and tumor recurrence. Developing imaging modalities to localize CSCs will help ensure CSCs eradication for a complete cancer cure. Discovery of more accurate yet biocompatible tracers, development of better techniques and higher imaging resolution and contrast are in progress. MRI, PET, and fluorescence imaging are the most promising modalities for clinical application of CSCs detection. Integration of all aspects of molecular imaging for CSCs detection will accelerate bench-to-bedside translation. Disclosure of conflict of interest The authors declare that they have no conflict of interest.

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Abbreviations CSCs, Cancer stem cells; AML, Acute Myeloid Leukemia; TSC, tissue stem cell; SP, side population; ALDH, Aldehyde Dehydrogenase; PET, Positron Emission Tomography; MRI, Magnetic Resonance Imaging; FMT, Fluorescence-Mediated Tomography; NIR, Near Infrared; mAb, monoclonal antibody; BCSCs, breast cancer stem cells; SPECT, Single Photon Emission Computed Tomography; USPIO, Ultrasmall Superparamagnetic Iron Oxide; SPIO, Superparamagnetic Iron Oxide; HA-MNCs, hyaluronan-modified magnetic nanoclusters. Address correspondence to: Yusri D Heryanto, Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa, Maebashi, Gunma 3718511, Japan. Tel: +81-27-220-8415; Fax: +81-27220-8409; E-mail: [email protected]; [email protected]

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